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Molecular Signatures in Sarcomas - The Translocation Paradigm

George T. Calvert, MD
Orthopaedic Oncology Fellow
Huntsman Cancer Institute & Primary Children’s Medical Center
University of Utah
Salt Lake City, Utah

Kevin B. Jones, MD
Attending Orthopaedic Oncologist
Huntsman Cancer Institute & Primary Children’s Medical Center
University of Utah
Salt Lake City, Utah

R. Lor Randall, MD, FACS
Director, Sarcoma Services
The L.B. & Olive S. Young Endowed Chair for Cancer Research Chief, SARC Lab
Huntsman Cancer Institute & Primary Children’s Medical Center
University of Utah
Salt Lake City, Utah

What is a translocation?

Translocations are genetic abnormalities caused by the rearrangement of non-homologous chromosomes.  Balanced translocations occur when there is no net loss of genetic material during the rearrangement.  Reciprocal translocations result when segments from non-homologous chromosomes are exchanged.  Non-reciprocal translocations involve the one-way transfer of genetic material from one non-homologous chromosome to another non-homologous chromosome.  A Robertsonian translocation occurs when the long arms of two acrocentric chromosomes are joined.  Acrocentric chromosomes (13, 14, 15, 21, and 22 in humans) are chromosomes with very short p arms1

Translocation nomenclature

Hundreds of translocations have been identified in association with cancer.  A standard nomenclature is used to describe translocations in order to prevent confusion.  The Committee for the International System of Cytogenetic Nomenclature (ISCN) publishes a guide to cytogenetic nomenclature.  A representative example is the most common Ewing sarcoma translocation t(11;22)(q24;q11.2-12).  The first set of parentheses indicates the two chromosomes involved in the translocation.  The second set of parentheses indicates the breakpoints on the respective chromosomes.  The letter p indicates the short arm, and the letter q indicates the long arm.  The numbers following the p or q denote the specific region and specific band of the involved arm. For example 11q24 is chromosome 11, long arm, region 2, band 4, and is appropriately stated "eleven q two four."

How do translocations cause cancers?

Despite the identification of hundreds of translocations related to cancer, all of the translocations analyzed to date cause pathology by one of two main mechanisms2.  The first mechanism is the creation of a fusion (sometimes called chimeric) protein.  The translocation places portions of the genes for two separate proteins next to each other and a new pathogenic gene product results.  The Philadelphia chromosome is an example of this mechanism, in which portions of the BCR and ABL genes are placed together, creating a new fusion product BCR/ABL.  This is the mechanism by which all studied sarcoma-related translocations function.  The second mechanism occurs when the translocation places a gene or genes in a new regulatory environment.  Burkitt lymphoma is a classic example of this second mechanism in which the MYC oncogene is up-regulated by being placed under the control of IGH gene regulatory elements.  Unlike the complex cytogenetic abnormalities seen in most solid organ cancers, translocation-associated sarcomas tend to have uniform chromosomal abnormalities from patient to patient and cell to cell.   

How are translocations identified?

During much of the cell cycle, genetic material is not bundled tightly into chromosomes, and it is not amenable to analysis of chromosomal structure.  During metaphase, the paired chromosomes are aligned, thus permitting structural analysis.  Using special stains on cells in metaphase, cytogeneticists are able to label the paired chromosomes.  The resulting karyotype is used to identify translocations.  Other techniques are also used, some more commonly in the clinical setting.  Florescent in situ hybridization (FISH) can label specific breakpoints in the genome, marking involved genes of interest.  Reverse-transcriptase polymerase chain reaction (RT-PCR) is used to detect the fused messenger RNA resulting from the aberrant joining of the two genes.  Flow cytometry can also be used in evaluating chromosomal abnormalities.

Which cancers involve translocations?

Cytogenetic analysis of tissue from most types of cancer reveals chromosomal abnormalities.  The cytogenetic abnormalities seen in many cancers come about during the later stages of the oncogenic process.  Increasing dysregulation of cell growth and division results in these late abnormalities.  This dysregulation often results in gross abnormalities which vary from individual to individual or even cell to cell within a tumor.  The chromosomal abnormalities found in these cancers are not often believed to be causative or central events in their pathogenesis.  Translocations are therefore believed to be the primary cause of only a subset of cancers.  A translocation which produces what is termed the Philadelphia chromosome t(9;22)(q34;q11) was the first genetic cause of cancer to be identified (1960).  It is found in 95% of chronic myelogenous leukemia cases.  Overall, translocations cause a greater percentage of leukemias and lymphomas than solid tumors.  However, among the solid tumors, translocations are causative or at least important factors in many sarcomas.  Table 1 lists sarcomas with known associated translocations.  Overall, these represent approximately one fourth of all sarcomas (Mertens, 2009).  The remaining sarcomas, such as osteosarcoma, chondrosarcoma, and pleiomorphic liposarcoma, have more complex cytogenic abnormalities. 

Table 1. Balanced Translocation Associated Sarcomas

Sarcoma

Translocation

5’ Fusion Partner

3’Fusion Partner

Alveolar rhabdomyosarcoma

t(2;13)(q35;q14)

PAX3

FKHR

Alveolar rhabdomyosarcoma

t(1;13)(p36;q14)

PAX7

FKHR

Alveolar soft part sarcoma

t(X;17)(p11.2;q25)

ASPL

TFE3

Angiomatoid fibrous histiocytoma

t(12;16)(q13;p11)

FUS

ATF1

Clear cell sarcoma

t(12;22)(q13;q12)

EWS

ATF1

Congenital fibrosarcoma/congenital mesoblastic nephroma

t(12;15)(p13;q25)

ETV6

NTRK3

Dermatofibrosarcoma protuberans

t(17;22)(q22;q13)

PDFGB

COL1A1

Desmoplastic small round cell tumor

t(11;22)(p13;q12)

EWS

WT1

Endometrial stromal sarcoma

t(7;17)(p15;q21)

JAZF1

JJAZ1

Ewing’s sarcoma/PNET

t(11;22)(q24;q12)

EWS

FLI1

Ewing’s sarcoma/PNET

t(21;22)(q22;q12)

EWS

ERG

Ewing’s sarcoma/PNET

t(7;22)(p22;q12)

EWS

ETV1

Ewing’s sarcoma/PNET

t(17;22)(q12;q12)

EWS

FEV

Ewing’s sarcoma/PNET

t(2;22)(q33;q12)

EWS

E1AF

Ewing’s sarcoma/PNET

t(16;21)(p11;q22)

FUS

ERG

Inflammatory myofibroblastic tumor

t(1;2)(q22;p23)

TPM3

ALK

Inflammatory myofibroblastic tumor

t(2;19)(p23;p13)

TPM4

ALK

Inflammatory myofibroblastic tumor

t(2;17)(p23;q23)

CLTC

ALK

Low grade fibromyxoid sarcoma

t(7;16)((q33;p11)

FUS

CREB3l2

Myxoid chondrosarcoma

t(9;22)(q22;q12)

EWS

CHN

Myxoid chondrosarcoma

t(9;15)(q22;q21)

TFC12

CHN

Myxoid chondrosarcoma

t(9;17)q22;q11)

TAF2N

CHN

Myxoid liposarcoma

t(12;16)(q13;p11)

TLS

CHOP

Myxoid liposarcoma

t(12;22)(q13;q12)

EWS

CHOP

Synovial sarcoma

t(X;18)(p11;q11)

SYT

SSX1

Synovial sarcoma

t(X;18)(p11;q11)

SYT

SSX2

Synovial sarcoma

t(X;18)(p11;q11)

SYT

SSX4

Adapted from Mercado GE, Barr FG. Chromosomal Translocations in Sarcomas: New Perspectives.3

Ewing sarcoma

The t(11;22)(q24;q12) translocation found in 85% of Ewing cases results in fusion of portions of the EWSR1 gene on chromosome 22 with portions of the FLI1 gene on chromosome 11.  The FLI1 gene is a transcription factor of the ETS family.  The function of the EWSR1 gene is presently unknown.  The EWS/FLI fusion protein functions as an aberrant transcription factor4.  EWS/FLI expression is required to maintain the oncologic phenotype of Ewing cell lines suggesting that it is a crucial oncoprotein in the disease.  Most of the other Ewing cases (approximately 15%) involve fusion of the EWS gene with a gene closely related to FLI. Many investigators are working to elucidate the pathway by which the Ewing's sarcoma fusion protein causes disease.  An interesting recent finding in this effort was that EWS/FLI acts upon sequences of repetitive DNA called microsatellites4.  These sequences are often otherwise colloquially referred to as “junk DNA” because they have long had no known function.  The discovery that microsatellites are involved in expression regulation by an oncogene suggests that these common genetic sequences may not be “junk” as previously thought.  Greater understanding of the EWS/FLI fusion oncogene may ultimately lead to new prognostic and therapeutic strategies.

Synovial sarcoma

The t(X;18)(p11;q11) translocation is detectable in nearly all synovial sarcomas.  The SYT gene (also called SS18), located on chromosome 18, is constitutively expressed in all human and mouse cells and is localized to the nucleus.  It is believed to be a transcriptional activator5.  The SSX gene family (of which SSX1, SSX2, and SSX4 are fusion partners in synovial sarcoma), located on the X chromosome, is believed to function by repressing target genes.  The SYT-SSX fusion protein usually contains most of the SYT gene but has the last 8 amino acids replaced by 78 amino acids from SSX5. The mechanism by which this fusion product causes cancer is just beginning to be understood.  A recent advance was the development of a synovial sarcoma mouse model using conditional gene expression.  Researchers created a mouse which expresses the SYT-SSX2 fusion protein selectively within skeletal muscle progenitor tissues.  These mice developed tumors with histology, immunohistochemistry, and transcriptional profile consistent with human synovial sarcoma5.  This model may aid in the elucidation of molecular pathways critical to synovial sarcoma genesis and in the analysis of new treatments for the disease.

Diagnostic utility of translocation research

Basic cytogenetic and molecular research has already proved useful in the treatment of patients.  The benefits of this translational research are most evident in the realm of diagnosis and prognosis of these rare malignancies.  There are greater than 50 distinct sarcoma subtypes.  Even with appropriate clinical history, physical examination, imaging and biopsy, accurate diagnosis can be challenging.  Clinicians and pathologists have been aided in this process by advances in cytogenetics and molecular biology.  Traditional cytogenetic analysis may fail to identify a translocation if the biopsied tumor cells fail to divide in culture.  This may occur due to the biology of the tumor itself or due to handling and processing of the specimen.  In addition to traditional cytogenetic analysis, immunohistochemistry stains, reverse transcriptase PCR (RT-PCR), and FISH analysis are available help determine the diagnosis of many translocation associated sarcomas.  Many of the available immunologic and molecular tests in clinical use are listed in Table 2.  An example of the utility of these tests is provided in Ewing sarcoma.  The Ewing sarcoma family of tumors can be difficult to distinguish from other small, round blue cell tumors based on histologic analysis alone.  RT-PCR and FISH are often used to confirm or refute the diagnosis.  Accurate diagnosis is of course crucial for determining the most appropriate treatment for patients.

Table 2.

Sarcoma

IHC

RT-PCR

FISH

Alveolar rhabdomyosarcoma

 

Y

Y

Alveolar soft part sarcoma

TFE 3

Y

 

Angiomatoid fibrous histiocytoma

 

 

Y

Clear cell sarcoma

 

Y

Y

Congenital fibrosarcoma/congenital mesoblastic nephroma

 

Y

Y

Dermatofibrosarcoma protuberans

 

Y

Y

Desmoplastic small round cell tumor

WT1

 

Y

Endometrial stromal sarcoma

 

Y

 

Ewing sarcoma/PNET

FLI1,O13/CD99

Y

Y

Inflammatory myofibroblastic tumor

ALK

Y

Y

Low grade fibromyxoid sarcoma

 

Y

Y

Extra skeletal myxoid chondrosarcoma

 

Y

Y

Myxoid liposarcoma

 

 

Y

Synovial sarcoma

TLE 1

Y

Y

Adapted from Jain S, Xu R, Prieto VG, Lee P.  Molecular Classification of Soft Tissue Sarcomas and Its Clinical Applications 6

Prognostic value of translocation research

Among patients with the same sarcoma type, the clinical behavior of the tumors can vary greatly.  Traditional prognostic factors such as tumor size, location, site, grade, and response to therapy remain useful for many sarcoma types.  However, molecular approaches to prognosis offer the potential for greater accuracy and earlier identification of the more aggressive sarcomas.  Presently, evidence suggests that molecular sub-typing of Ewing, synovial, and alveolar rhabdomyosarcoma may have prognostic value2.  Ewing sarcoma patients with subtypes other than the common EWS-FLI1 have historically had worse prognoses; however, this difference has diminished with current chemotherapy protocols. More recently, researchers have identified members of the glutathione S transferase (GST) family as downstream targets of the Ewing fusion protein.  Levels of GSTM4 and MGST1 were shown to inversely correlate with survival among patients with Ewing sarcoma4.  GST research is presently investigational and is not yet used for prognostic testing in standard clinical situations.   Synovial sarcoma patients with the SSX1 variant had worse survival in some but not all studies5.  Among alveolar rhabdomyosarcoma patients, the PAX3 variant of the fusion protein appears to be more aggressive2.

Another molecular technique which may ultimately provide diagnostic and prognostic information for sarcoma patients is complimentary DNA (cDNA) microarray technology.  This technique uses mRNA retrieved from tumors specimens to create a cDNA library which can then be analyzed to determine the relative levels of mRNA expression of different genes in the specimen.  This information provides a “molecular signature” of the individual tumor.  Although not used presently in routine clinical practice, this technique may ultimately provide prognostic information at the individual level and permit treatment tailored to the specific tumor7.

Treatment implications of translocation molecular research

Sarcoma translocations and their associated fusion proteins provide attractive targets for therapeutic research.  The translocations are uniformly expressed in cancer cells but not in normal cells.  This specificity provides an opportunity for selectively targeting the cancer cells without harming normal cells.  Possible therapeutic strategies include development of antibodies or small molecules which can block the function of the fusion protein.  Another strategy is development of antisense oligonucleotides capable of directly blocking the mRNA transcript of the fusion gene. 

For sarcomas, these strategies remain experimental with numerous compounds in development at the animal testing or phase 1 clinical trial stage.  Selective treatment of translocation-associated cancers has progressed further among leukemias and lymphomas.  Imatinib (Gleevec in the US; Glivec in Europe) was approved by the U.S. Food and Drug Administration (FDA) for treatment of CML in 2001.  It is now a first-line treatment for CML and several other cancers that involve the tyrosine kinase pathway.  Imatinib was developed by a rational drug design effort which sought to identify compounds which selectively inhibit the function of the BCR/ABL fusion protein discussed previously.  The ABL1 gene encodes a tyrosine kinase, which becomes constitutively active when fused with BCR.  Imatinib has some binding affinity for other tyrosine kinases and is therefore not completely selective.  Additionally, cancer cells may become resistant to imatinib through a variety of mechanisms.  Second-generation tyrosine kinase inhibitors are now under development to address these issues. 

Fundamental understanding of the molecular biology of sarcomas lags behind that of CML because the Philadelphia chromosome (and the BCR/ABL gene fusion) was discovered earlier, has been more extensively studied, and is associated with a disease much more common in the population.  The translation of laboratory discoveries to selective clinical treatments for leukemias suggests that the prospects for similar treatments of sarcomas are good.  Investigators are actively studying many of the more promising agents.

References

  1. O'Connor, C. (2008) Human chromosome translocations and cancer. Nature Education.  Retrived April 2011 from http://www.nature.com/scitable/topicpage/human-chromosome-translocations-and-cancer-23487
  2. Mertens F, Antonescu CR, Hohenberger P, Ladanyi M, Modena P, D'Incalci M, Casali PG, Aglietta M, Alvegård T. Translocation-related sarcomas. Semin Oncol. 2009 Aug;36(4):312-23.
  3. Mercado GE, Barr FG. Chromosomal Translocations in Sarcomas: New Perspectives. Electronic Sarcoma Update Newsletter.  Retrieved April 2011 from http://sarcomahelp.org/research_center/chromosomal_translocations.html
    #Toomey EC, Schiffman JD, Lessnick SL. Recent advances in the molecular pathogenesis of Ewing's sarcoma. Oncogene. 2010 Aug 12;29(32):4504-16.
  4. Haldar M, Randall RL, Capecchi MR. Synovial sarcoma: from genetics to genetic-based animal modeling. Clin Orthop Relat Res. 2008 Sep;466(9):2156-67.
  5. Jain S, Xu R, Prieto VG, Lee P. Molecular classification of soft tissue sarcomas and its clinical applications. Int J Clin Exp Pathol. 2010 Apr 23;3(4):416-28.
  6. Randall RL, Wade M, Albritton KH, Coffin CM, Joyner DE. Validation of cDNA microarray analysis to distinguish tumor type ex vivo. Clin Orthop Relat Res. 2003 Oct;(415 Suppl):S110-9.

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